Neurofilaments (NF) are characteristic structures in normal neurons that become abnormally prominent and distributed, as well as chemically altered, in certain spontaneous neurological disorders and after accidental or experimental insult. Neither the mechanism by which these alterations occur nor the function of NF in normal neurons, are well understood. The objectives of this project are to gain insight into these issues by analyzing the metabolism of the three protein subunits comprising NFs (NF-L, NF-M and NF-H) in normal neurons and examining the consequences of alterations in their metabolism. The specific aims focus on the major chemical modification of NF proteins, phosphorylation, and on the enzymes that catalyze the addition (protein kinases) and removal (phosphatases) of phosphate groups. Primary cultures of pure neurons are used as a model system, as they allow the examination of the activities of these enzymes in intact cells and also permit perturbation by exogenous agents and conditions. Pervious work has located at least 15 phospho-acceptor sites in the carboxyterminal tail domain of NF-M, and has implicated two types of kinases that are involved in phosphorylating different subsets of these sites. A kinase that phosphorylates one subset of sites, a NF-associated kinase (NFAK), has been partially purified and has properties similar to casein kinase I. Further detailed analysis of this kinase is planned, including the production of specific antibodies, cloning the cDNA, and identification of exact phosphate acceptor sites. Analysis of the expression of this enzyme will be carried out at both the protein and mRNA levels. Distribution of the kinase within different neuronal intracellular compartments will be determined by immunological methods. The in vivo consequences of suppressing expression of NFAK will be examined by exposure to antisense oligonucleotides. Other experiments will address the role of phosphatases in regulating the integrity of NF. Lastly, the effects of neurotoxins on NF organization and phosphorylation, as well as on the expression and activity of the kinases and phosphatases, will be investigated.